Oligonucleotide capture during end joining in mammalian cells.

AUTOR(ES)

Roth, D B

RESUMO

Extra nucleotides (termed filler DNA) are found at about 10% of the junctions of the genetic rearrangements that arise by illegitimate recombination in mammalian cells. Such filler DNAs could arise by the joining of oligonucleotide fragments to broken ends prior to end joining. We tested this possibility by microinjecting mixtures of defined oligonucleotides with SV40 genomes that were linearized in the intron for T antigen, a site where incorporation of extra nucleotides does not impair viability. Using an injection ratio of 1000 oligonucleotides per DNA end, we screened viable genomes for incorporation of single-stranded and double-stranded oligonucleotides with varying degrees of complementarity to the ends of the linear SV40 molecules. Genomes from 510 independent plaques were screened by restriction digestion to identify those that had picked up a restriction site unique to the injected oligonucleotides. Double-stranded oligonucleotides that were fully complementary to the SV40 ends were readily incorporated, but uptake of the other oligonucleotides was not detected by restriction analysis. Nucleotide sequences of junctions from 12 genomes derived from co-injection of noncomplementary oligonucleotides revealed two with filler DNA, but neither could be assigned unambiguously to the injected oligonucleotides.

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